Experimental Cell Research 89 (1974) 343-351 ... - Science Direct

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Experimental Cell Research 89 (1974) 343-351

SEPARATION

OF LYMPHOID-LINE

CELLS

AND

ACCORDING

TO VOLUME

DENSITY

TH. A. W. SPLlNTERl

and M. REISS

Laboratory of the Netherlands’ Red Cross Blood Transfusion Service, and Laboratory of Experimental and Clinical Immunology, Amsterdam, The Netherlands

Central

University

SUMMARY Velocity sedimentation and isopycnic centrifugation was used to separate exponentially growing RAJI lymphoid line cells according to size and density. Mixtures of Ficoll, Isopaque and tissue culture medium were used as gradient media. These media had a constant pH, were isotonic, and did not have any significant harmful effect on the cells. The observed variation in cell size paralleled the progression of the cells through the cell cycle, as assessedby thymidine incorporation and impulse cytophotometric determination of DNA contents. Differences in cell density did not reflect the cell cycle phase. No correlation could be established between cell size and density. Velocity sedimentation could be used to obtain cell populations which were relatively pure according to cell cycle phase and growing synchronously for at least 24 h.

When blood lymphocytes are stimulated by an antigen, or non-specific mitogen or allogeneic cells in vitro, part of the cells enter the cell cycle from their resting or GO phase [I]. Only part of these cells go into mitosis [2]. After stimulation some have been described to be capable of producing immunoglobulins [3], of mediators of cellular immunity [4], or exhibit a cytotoxic effect on target cells [5] depending on the nature of stimulation and the type of stimulated cells. Some of these characteristics may be limited to a special cell cycle phase as has been shown in lymphoid cell lines for the production of immunoglobulins [6] and migration inhibitory factor [7]. In order to gain insight into these different processes and to be able to perform kinetic 1 Postal address: Plesmanlaan 125, P.O. Box 9190, Amsterdam, The Netherlands. 23-741802

studies on the development of lymphoid cells wjth a particular function we applied sedimentation techniques to separate lymphoid line cells according to their cell cycle phase. This method was preferred since other synchronization procedures always alter normal cell metabolism and produce only a limited degree (60-70%) of synchrony [8, 91. We have chosen lymphoid line cells as an experimental model to evaluate various separation procedures. These cells have the following advantages: they have several characteristics in common with activated lymphocytes [lo], have constant growth characteristics and large numbers of cells are available for study at any time. We made use of the velocity sedimentation technique for selection of cells according to cell size and of isopycnic centrifugation for separation according to cell density. The latter method has been reported recently to Exptl Cell Res 89 (1974)

344 Splinter and Reiss Table 1. Composition and properties of the Ficoll-lsopaque solutions

Polysucrose (Ficoll) 1.156 M metrizoate (Isopaque) 0.175 M Tris-HCl (pH 7.4 at 0°C) Distilled water Density at 4°C Refractive index at 25°C Osmolarity pH at 4°C

High density solution

Low density solution

250.0 g 80.0 ml 143.0 ml 834.0 ml 1.100 g/ml 1.3712 288-300 mOsm 7.4

30.0 g 118.0 ml 143.0 ml 745.0 ml I .064 g/ml 1.3515 288-300 mOsm 7.4

The undiluted iFopaque solution as is used for the preparation of the solutions in table 1 has a concentration of 1.156 M metrizoate. The same solution was used by Loos and Roos (Exptl cell res 86 (1974) 334), but in that article a concentration of 0.156 M instead of 1.156 metrizoate was wrongly given.

separate activated from resting blood lymphocytes [ 111. The technique of isopycnic centrifugation has been described recently by Loos & Roos [12], the velocity sedimentation by Phillips [13], and others [14, 151. A number of investigators have employed this technique to separate cultured cells according to cell cycle phase [14, 151. Generally, these authors neither mention the properties of their gradient materials, such as pH and osmolarity, nor the effects of the separation procedure on the quality of the cells. Therefore, we have investigated these possible harmful effects on the lymphoid line cells. Moreover, we have compared velocity sedimentation and isopycnic centrifugation as techniques to separate cells according to their cell cycle phase.

MATERIALS

AND METHODS

Chemicals Ficoll (polysucrose, mol. wt 400 000) was obtained from Pharmacia, Uppsala, Sweden, and Isopaque (sodium-, calcium and magnesium-N-methyl-3,5diacetamido 2,4,6-triiodobenzoate) from Nyegaard & Co., Oslo, Norway. RPMI-1640 was purchased from Flow Laboratories Irvine, (Ayrshire, Scotland), fetal calf serum (FCS) from GIBCo (Grand Island, N.Y.), and glutamine from Merck (Darmstadt, GFR). 3HMethyl-thymidine (spec. act. 17 Ci/mmole) was obtained from the Radiochemical Centre (Amersham, UK). Exptl Cell Res 89 (1974)

Ceil culture An exponentially growing RAJI lymphoid cell line, derived from cells from a patient with Burkitt’s lymphoma, was employed. Cells were grown at 37”C, in suspensions of 100 ml in glass roller bottles ( ~9 cm; height 20 cm, total volume 700 ml), revolving at a speed of 5 rpm. Alternatively, smaller volumes of 3&50 ml were cultured in 250 ml Falcon tissue culture flasks (no. 3024). The growth medium consisted of 80 % (v/v) RPM1 1640, and 20% (v/v) fetal calf serum (FCS), supplemented with glutamine (0.03 % w/v), penicillin (100 W/ml) and streptomycin (100 pg/ml). Cells were maintained at a concentration of 2-10 x lo6 cells/ml. This was achieved by splitting and feeding every 48-72 h. A Coulter counter (model ZF) was used for daily electronic cell counting.

Cell harvesting Cells were harvested by centrifugation at room temperature for 5 min, at 60 g,,,. Medium was then added to the pellet, in order to obtain a suspension with a final concentration of 7-10 X 106cells/ml.

Gradients For both velocity sedimentation and isopycnic centrifugation, linear density gradients were prepared from two sterile stock solutions, by a modification of the method described by Loos & Roos [12]. The procedure was performed as follows. Two solutions of Ficoll-&opaque were prepared, with a density of 1.064 and 1.100 g/ml respectively. The composition and properties of these mixtures are given in table 1. To these solutions different amounts of culture medium (d = 1.012 g/ml) were added to obtain stock solutions of the required densities (see table 2). Densities were measured at 4°C with a Mohr’s balance. Densities of gradient fractions were checked by measuring the refractive index at 25°C with an Abbe refractometer (type no. 1721, Carl Zeiss, Oberkochen, GFR). The final solutions contained 6-17 % (v/v) FCS, and their properties as to osmolarity and pH were similar to the original Ficoll-Isopaque mixtures. Gradients for the velocity sedimentation were pre-

Separation of LLC

345

Table 2. Composition of stock solutions for the gradients Required density (g/ml) Components

1.020

1.040

1.045

1.074

Ficoll-Isopaque d= 1.100 g/ml Ficoll-Isopaque d = 1.064 g/ml Culture medium d- 1.012 g/ml

-

-

-

6.95 ml

1.54

5.40 ml

6.35 ml

-

8.46 ml

4.60 ml

3.65 ml

3.05 ml

pared in the following manner: first a 3 ml layer of the high-density Ficoll-Isopaque solution (d- 1.100 g/ml) was placed at the bottom of a glass Kimax tube (02.86 cm, height 12.25 cm, total volume 54 ml). On top of this layer, the linear gradient was pumped [12]. At the start of the procedure, the mixing chamber and the stock tube contained 20 ml of the lighter and the heavier solutions respectively. Then the heavier stock solution (d= 1.054 g/ml) was pumped into the mixing chamber at a rate of 0.6 ml/ min. From the mixing chamber the gradient material was pumped into the Kimax tube at 1.2 ml/min. This procedure took 35 min and the obtained gradient had a height of approx. 80 mm. Finally, a layer of 3 ml of culture medium was added, to act as a destreamer. A cell suspension of 3 ml, containing 2030 x lo6 cells was layered on top of the destreamer shortly before centrifugation. For the isopycnic centrifugation the gradients were prepared in a similar manner. In this case stock solutions with densities of 1.040 g/ml and 1.074 g/ml were employed. However, the bottom layer of heavy Ficoll-Isopaque and the destreamer layer were omitted. In the experiments in which the cells were recultured after separation, the procedure took place under aseptic conditions in a laminar flow cabinet.

Size and density profiles Centrifugation was carried out in a centrifuge with a stable rotor at 4°C during 15 min at 25 g,,, for the velocity sedimentation and at 2 400 g,,, for the isopycnic centrifugation. Afterwards, a long steel needle was carefully lowered to the bottom of the tube and the material was pumped out in fractions of 1.8 ml. The ceils were counted in each fraction electronically and the densities were checked by measuring the refractive index in a number of fractions at 25°C after spinning down the cells (Eppendorf centrifuge, model no. 3200, 2 min at room temperature).

Precursor incorporation For the analysis of the distribution of DNA-synthesizing cells, suspensions were preincubated at 37°C for 2 h with 3H-TdR (final cont. 7.2 pg/ml, 3 &i/ml) in growth medium. Cell number was 5-10 x lo5 cells/ml.

Uptake of SH-TdR was stopped by adding approx. 2 mg of unlabelled thymidine and storing the samples at 0°C. The cells were then collected on glass fibre filters, washed carefully with distilled water, dried, and dissolved in 1 ml soluene 350 (Packard Instruments Company, Downers Grove, Ill.) A toluenebased scintillation mixture was added, and the radioactivity was determined in a Packard Tri Carb liquid scintillation counter (model 3320).

Distribution

of DNA contents of cells

Profiles of DNA content/cell were obtained by means of a Phywe ICP 11 impulse cytophotometer (ICP) as described by Smets 1171.In short, the cells were fixed in alcohol (96 % v/v) and treated with RNAase, and pepsine to remove RNA and protein. Afterwards, DNA contents were measured in a suspension of the remaining nuclei suspended in buffer containing 10 pug/ml ethidium bromide, and the profiles were recorded.

RESULTS All experiments were at least once repeated. The results were virtually similar to each other. Effects of the separation procedures on the cells The influence of the separation procedures on cell characteristics was assessed in several ways. The average yield of cells after velocity sedimentation was 80 46 (SD. 10.5 %, n = I I), after isopycnic centrifugation 85 % (S.D. 12.3 ‘$6, n= 11). However, when a Fico&Isopaque gradient was used without the addition of FCS the yield was 50 and 60%, respectively. Cell viability, as determined by Exptl Cell Res 89 (1974)

346

Splinter and Reiss logy of the cells after centrifugation was excellent. We concluded that the separation procedure was harmless enough to warrant further experimentation.

b

Size and density profiles of exponentially growing cultures The density of asynchronous RAJI cells ranged from approx. 1.045 g/ml to 1.070 g/ ml, with a mean of 1.057 g/ml (fig. 1a). The volume distribution of these cells is asymmetrical (fig. %a), in accordance with the curve obtained electronically by Crissman & Steinkamp [16]. If the velocity gradient was loaded with more than 30 x lo6 cells the distribution curve showed an increased recovery of cells in the bottom fractions. Therefore, we decided that 30 x lo6 cells was the limit for this gradient. Relationship between cell size and density

Fig. 1. (a) Abscissa: (a) density (g/ml); (b) fraction no.; ordinate: (a, b) no. of lymphoid cells/fraction (% of total recovered cells). aLA, Light cells (1.04X 1.056 g/ml); l . . .o, heavy cells (1.056-1.070 g/ml); 0-0, entire population. Density centrifugation and subsequent velocity sedimentation of light and heavy cells (see text).

trypan blue exclusion, was 90 y0 average after the procedures, as compared to 95-99s in growing cultures. Any dead cells present were retained in the first two fractions at the top of the velocity gradient or in the last two fractions at the bottom of the density gradient. Cells which were brought back into culture after centrifugation (see below) had the same doubling time (24 h) as the asynchronous culture from which they had been obtained. Contact with the gradient material did not influence the cell size, but the cell density appeared to increase somewhat during centrifugation. The separation procedure had no significant effect on the 3H-TdR incorporation of cells (Student’s t test: p>O.20; n = 32). Moreover, stainability and marphaExptl Cell Res 89 (1974)

In order to determine whether a correlation between size and density could be found, we performed the following two experiments. In the first one, two identical density gradients were loaded with one-half of a cell suspension each. After centrifugation, the material of one tube was pumped out in small fractions as described under Methods. From the other tube two pooled fractions were obtained of light and heavy cells respectively (fig. 1 a). These latter suspensions were washed once with phosphate-buffered saline (PBS), and centrifuged at room temperature during 5 min at 60 g,,,. The pellets were resuspended in culture medium and then separated by velocity sedimentation. Fig. 1b shows the results: the volume distribution of light and heavy cells was essentially the same as that of unseparated cells. In the second experiment, the opposite procedure was followed: the cells were separated into a fraction with large and a fraction with small cells, and both were sub-

Separation of LLC jetted to isopycnic centrifugation (fig. 2). Larger and smaller cells showed no difference in their densities. However, the mean density had increased from 1.057 to 1.060 g/ ml, as compared with the control cells which had only been centrifuged once. This difference almost disappeared if the light and heavy fractions were incubated in growth medium at 37°C for 30 min before the density centrifugation. Thus, in these experiments no correlation could be found between cell volume and density, but Ficoll-Isopaque seems to induce a reversible increase in cell density. Separation ojpre-labeled

cells

Our next step involved the demonstration of changes in cell volume and density during

lo-

Fig. 2. Abscissa:(a) fraction no.; (b) density (g/ml); ordinate: no. of lymphoid cells/fraction (% of total

recovered cells). A-A, Small cells (fraction l-10); l . . .O, large cells (fraction 11-22); O-0, entire population. Velocity sedimentation and subsequent density

centrifugation of large and small cells (see text).

341

Fig. 3. Abscissa: fraction no.; ordinate:

no. of cells and incorporated SH-TdR ner fraction (both as % of total recovery). 0-0, Ceil no.; 0.. . O,‘incorporated 3H-TdR. Velocity sedimentation of asynchronous cells prelabelled with SH-TdR (see text).

part of the cell cycle. The position of DNAsynthesizing cells (=S phase cells) in size and density profiles was assessed by labelling the cells before separation. A suspension of asynchronously growing cells in culture medium was incubated with 3H-TdR during 2 h as described under Methods. After the incorporation was stopped, the cells were spun down and resuspended in a small volume of medium. These suspensions were then subjected to velocity and density centrifugation. The cells were counted and the radioactivity was determined in each fraction as described above. The radioactivity in every fraction was recorded as percentage of the entire radioactivity that was recovered. The same was done for the cell number. Figs 3 and 4 show the results. Cells which had incorporated 3H-TdR were recovered mainly in the middle portion of the volume profile. The density distribution of the labelled cells was essentially similar to the density profile of the population as a whole. Therefore, the cell volume changes as the cells synthesize DNA, whereas the specific gravity seems to remain constant. Exptl Cell Res 89 (1974)

348

Splinter and Reiss

4’ :

20-

n : ‘! I

i' i

15.

:

lo-

:

~

1

medium-sized cells represent cells in the G 1 and S phase, respectively. The largest particles consisted mainly of G2 and M phase cells, together with cells with a lower DNA content. The reduced purity in this fraction was probably due to random clumping. The pools of cells of low, medium, and highdensity displayed ICP recordings as shown in fig. 6. All phases of the cell cycle were present in each pool. Some enrichment of the medium and high density pools with S and G2 phase cells seemed to be present. This is not in complete agreement with the previous experiment, but might be explained by the observed influence of Ficoll-Isopaque on cell density, as described earlier. However, these experiments clearly demonstrate that cells can be separated according to their cell cycle phase by velocity sedimentation but not by density centrifugation.

:L,,/+$-%+j ,+i.&& 1040

1050

1060

Fig. 4. Abscissa: density (g/ml); ordinate: no. of cells and incorporated aH-TdR per fraction (both as % of total recovery). o-o, Cell no.; l . . .O, incorporated SH-TdR. Density centrifugation of asynchronous cells, prelabelled with 3H-TdR (see text).

Relationship between cell size and density, and DNA content The DNA contents of cells of different size and density were determined by means of impulse cytophotometry as described under Methods. Exponentially growing cells were subjected to velocity or density centrifugation. The gradients were then fractionated in the usual manner and cell counts determined. Next, several fractions of each gradient were pooled into three portions as indicated in figs 5 and 6. The choice of the pools from the velocity gradient was determined by the distribution of labelled cells as indicated by the arrows in fig. 3, the pools from the density gradient were chosen arbitrarily. In each pool DNA contents were measured. DNA contents range from 2N (pure Gl phase cells) to 4N (G2 and M phase cells). S phase cells with varying degrees of completion of DNA replication are distributed between 2 and 4N. Because the surface area of the different ICP recordings are not identical, they are not comparable to each other. Fig. 5 clearly shows that the small and Exptl Cell Res 89 (1974)

Fig. 5. Abscissa: (a) fraction no.; (b) DNA content in terms of the haploid number of chromosomes (N); ordinate: (a) no. of cells/fraction ( % of total recovered cells); (6) no cells counted per channel. O-O, Cell no. (velocity sedimentation); -, cell no. (DNA profile). DNA content/cell in suspensions of small, rnediumsized and large cells as obtained by velocity sedimentation (see text).

Separation of LLC

Fig. 6. Abscissa: (a) density (g/ml); (6) DNA content in terms of the haploid number of chromosomes; ordinate: (a) no. of cells/fraction (Y/oof total recovered cells); (b) no. of cells/channel. DNA contents of light, middle weight and heavy cells, as obtained by isopycnic centrifugation (see text).

Synchronous cultures Finally, we attempted to use the velocity sedimentation technique to obtain cell populations growing with a high degree of synchrony, as determined by ICP analysis. The entire procedure was carried out aseptically in a laminar flow cabinet. A suspension of approx. IO* cells in 12 ml growth medium was prepared from an asynchronous population as described under Methods. The medium in which the cells had been cultured in the previous 24 h was collected and stored at 37°C for subsequent use (see below). Four identical gradients for velocity sedimentation were loaded with 3 ml of the cell suspension each (maximally 30 x IO6 cells/tube), After centrifugation the gradients were fractionated according to the following scheme: from the first three tubes, the small and medium sized cells were collected as described above. The cells of the fourth tube were pooled as a whole. These three pools of cells were washed

349

once with culture medium and then incubated in prewarmed culture medium for 30 min at 37°C to reverse the effects of FicollIsopaque as described above. Afterwards, the cells were resuspended in 50 ml of their own preconditioned culture medium at a density of approx. 5 x lo5 cells/ml and brought back into culture in Falcon tissue culture flasks. At fixed intervals cells were counted and samples of 5 ml were drawn for ICP analysis of the DNA-content. Fig. 7 displays the ICP recordings of the three suspensions at 0, 24 and 48 h, as compared to a normal exponentially growing control suspension. The ‘recombined’ suspension of cells displayed a profile which 10

a recomblned

untreated 5

0 LYl!zlm 2N

2N

4N

4N

2N

4N

2N

4N

?N

4N

2N

4N

2N

4N

2N

4N

Fig. 7. Abscissa: DNA contents in terms of the haploid number of chromosomes; ordinate: no. of cells/ channel. (a) control suspensions; (6) “G 1” phase

cells; (c) “S” phase cells. DNA profiles in synchronous growing cells as obtained by velocity sedimentation. Samples taken at 0, 24 and 48 h after reculturing, Control suspensions: an asynchronous culture labelled ‘untreated’ and asynchronous cells that underwent the separation procedure but were pooled afterwards labelled ‘recombined’ (see text). Exptl Cell Res 89 1974

350

Splinter and Reiss

was identical with the control and had the same growth rate (doubling time: 24 h). The suspension of small cells contained almost pure Gl phase cells. After 24 h we found a population of cells in G2 and Gl phase and hardly any S phase cells. This is in agreement with the fact that the cell number was almost doubled, and indicated that almost all cells had completed one cycle and were still growing synchronously. After 48 h the ICP-recording showed an almost normal asynchronous profile. The suspension of medium-sized cells consisted mainly of S phase cells. After 24 h a population of cells in G 1 and early S phase was found and the cell number was doubled. This indicated that all cells had completed one cycle and were still growing synchronously. In this case also the synchronous pattern had nearly disappeared after 48 h. In summary, separation of exponentially growing cells by velocity sedimentation yielded populations with a high degree of synchrony, which was retained in culture during at least 24 h. DISCUSSION The technique of separation of lymphoid line cells according to size and density, as described above, is a relatively simple and fast procedure which is harmless to the cells. The latter property was assessed by morphological examination, trypan blue exclusion, 3HTdR incorporation, and growth rate after reculturing of separated cells. Furthermore, this method gave an acceptable yield of 8085 % of the cells if fetal calf serum was added to the Ficoll-Isopaque mixture. The physical changes which occur in cells progressing through a cell cycle, studied by the combined analysis of the DNA content, 3H-TdR incorporation, velocity sedimentation and density centrifugation enabled us to detect whether these changes were due to Exptl Cell Res 89 (1974)

the cell cycle or caused by the analytical procedure itself. Thus, we found that three possibilities exist in which artefacts may be introduced by our method. In the first place we found that isopycnic as well as velocity sedimentation induced a reversible increase in cell density. Secondly, when lymphoid line cells were synchronized in the Gl phase by medium exhaustion, an increase in the cell density was observed which was not related to the cell cycle. This was shown by analysis of the density distribution of these cells after pulse-labelling with 3H-TdR. We found that also the labelled cells (contaminating S phase cells) had increased in density. Moreover, analysis of the volume distribution by velocity sedimentation revealed a shift towards the region of small cells (unpublished observations). We interpret these results as physical changes due to damage by the synchronization and/or separation procedure. Experiments of this kind should therefore always include a control run with 3HTdR-labelled cells. Thirdly, ICP analysis of the fastest sedimenting fraction in a velocity gradient showed a cell population which was heterogeneous as to the cell cycle phase. As was suggested by Everson et al. [15], this may be due to contamination of the large cells with clumped smaller cells. Analysis of the density distribution of 3HTdR-labelled lymphoid line cells revealed a uniform density distribution of labelled cells in all density regions. Similar results were mentioned by MacDonald & Miller [14] for mouse L cells. In addition, we found an identical distribution of the DNA content at different cellular densities and no differences in the volume distribution of these cells. Moreover, the reverse experiment showed no differences in the density distribution of cells

Separation

of UC

351

possible correlations between the uptake of with different volumes. Thus we conclude a labelled compound and the cell cycle, as that no correlation exists between volume and density of human lymphoid line cells. shown with 3H-thymidine. On the other hand, we have found a clear We are much indebted to Dr J. A. Loos and Dr D. correlation between cell volume and cell Roos for their advice and to Dr H. K. Prim for his criticism. We also Gish to thank Dr L. A. Smets cycle phase, meaning that the progression of (National Cancer Institute, Amsterdam) for the imthese cells through the cell cycle is only ac- pulse cytophotometric measurements he performed us. The RAJI cell line was obtained from Dr G. companied by changes in cell volume but not for Klein, Stockholm. This investigation was financially supported by the by changes in cell density. This last concluNetherlands’ Organization for the Advancement of sion is remarkable in the light of the findPure Research (Z.W.O.), The Hague, The Netherings by Loos & Roos [II], who showed a lands (grant no. 91-5). clear shift in density after antigenic stimulation of human blood lymphocytes. REFERENCES The changes in physical properties which 1. Polgar, P R, Kibrick, S & Foster, J M, Nature cells undergo in the transition from GO to 218 (1968) 596. 2. Polgar, P R & Kibrick, S, Nature 225 (1970) 857. Gl phase may be essentially different from 3. Greaves, M & Janossy, G, Transpl rev 11 (1972) those found when cells progress through the 87. 4. David, J R & David, R R, Progress in allergy rest of the cell cycle. 16 (1972) 300. Finally, we have confirmed the findings of 5. Solliday, S & Bach, F H, Science 170 (1970) 1406. Everson et al. [15] that relatively pure pop6. Buell, D N & Fahey, J L, Science 164 (I 969) 1524. ulations of G 1 and S phase cells may be 7. Tubergen, D G, Feldmann, J D, Pollock, E M & Lerner. R A. J exvtl med 135 (1972) 255. obtained by velocity sedimentation. Brought 8. Calavazi,‘G, &he&, H & Bodtsma: D, Exptl back in culture, these cells grow synchroncell res 41 (1966) 428. 9. Drewinko, B, Lichtiger, B & Trujillo, J M, Bioously for at least 24 h. This procedure enmedicine 18 (1973) 30. ables us to perform kinetic studies on the cor10. Douglas, S b, Borjeson, J & Chessin, L N, J (1967) 340. relation between cell function and cell cycle 11. immuno199 Loos, J A & Robs, D, Exptl cell res 86 (1974) phase. 333. Ibid 86 (1974) 342. If a large number of cells in a special phase 12. -Miller, R G & Phillips, R A, J cell physiol 73 13. of the cell cycle is needed, e.g. the transition (1969) 191. 14. MacDonald, H R & Miller, R G, Biophys j 10 of Gl to S phase, the velocity sedimenta(1970) 834. tion technique is not suitable. However, it 15. Ever&n, L K, Buell, D N & Rogentine, G N, J exvtl med 137 (1973) 343. may be used to increase the degree of syn16. Crissman, H A h Stejnkamp, J A, J cell biol 59 chrony reached by chemical methods. Such (1973) 766. 17. Smets, L A, Nature new biol 239 (1972) 123. investigations are now in progress. The procedure may also be very suitable to screen Received May 29, 1974

Exptl Cell Res 89 (1974)